Capsule array devices and methods of use

ABSTRACT

This disclosure provides microwell capsule array devices. The microwell capsule array devices are generally capable of performing one or more sample preparation operations. Such sample preparation operations may be used as a prelude to one more or more analysis operations. For example, a device of this disclosure can achieve physical partitioning and discrete mixing of samples with unique molecular identifiers within a single unit in preparation for various analysis operations. The device may be useful in a variety of applications and most notably nucleic-acid-based sequencing, detection and quantification of gene expression and single-cell analysis.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/683,192, filed Aug. 14, 2012; U.S. Provisional PatentApplication No. 61/737,374, filed Dec. 14, 2012; U.S. Provisional PatentApplication No. 61/762,435, filed Feb. 8, 2013; U.S. Provisional PatentApplication No. 61/800,223, filed Mar. 15, 2013; U.S. Provisional PatentApplication No. 61/840,403, filed Jun. 27, 2013; and U.S. ProvisionalPatent Application No. 61/844,804, filed Jul. 10, 2013, whichapplications are incorporated herein by reference in their entiretiesfor all purposes.

BACKGROUND OF THE INVENTION

The detection and quantification of analytes is important for molecularbiology and medical applications such as diagnostics. Genetic testing isparticularly useful for a number of diagnostic methods. For example,disorders that are caused by mutations, such as cancer, may be detectedor more accurately characterized with DNA sequence information.

Appropriate sample preparation is often needed prior to conducting amolecular reaction such as a sequencing reaction. A starting sample maybe a biological sample such as a collection of cells, tissue, or nucleicacids. When the starting material is cells or tissue, the sample mayneed to be lysed or otherwise manipulated in order to permit theextraction of molecules such as DNA. Sample preparation may also involvefragmenting molecules, isolating molecules, and/or attaching uniqueidentifiers to particular fragments of molecules, among other actions.There is a need in the art for improved methods and devices forpreparing samples prior to downstream applications.

SUMMARY OF THE INVENTION

This disclosure provides compositions and methods for a microcapsulearray device.

An aspect of the disclosure provides a composition comprising a firstmicrocapsule, wherein: the first microcapsule is degradable upon theapplication of a stimulus to the first microcapsule; and the firstmicrocapsule comprises an oligonucleotide barcode. In some cases, thefirst microcapsule may comprise a chemical cross-linker. The chemicalcross-linker, for example, may be a disulfide bond. In some cases, thecomposition may comprise a polymer gel, such as, for example apolyacrylamide gel. The first microcapsule may comprise a bead. In somecases, the bead may be a gel bead.

Moreover, the stimulus may be selected from the group consisting of abiological, chemical, thermal, electrical, magnetic, or photo stimulus,and combination thereof. In some cases, the chemical stimulus may beselected from the group consisting of a change in pH, a change in ionconcentration, and a reducing agent. The reducing agent may be, forexample, dithiothreitol (DTT) or tris(2-carboxyethyl) phosphine (TCEP).

A second microcapsule may comprise the first microcapsule. Moreover, thesecond microcapsule may be a droplet. In some cases, the composition mayalso comprise a nucleic acid that comprises the oligonucleotide barcode,wherein the nucleic acid comprises a deoxyuridine triphosphate (dUTP).In some cases, the composition may comprise a polymerase unable toaccept a deoxyuridine triphosphate (dUTP). Also, the composition maycomprise a target analyte, such as, for example, a nucleic acid. Thenucleic acid may be selected from the group consisting of DNA, RNA,dNTPs, ddNTPs, amplicons, synthetic nucleotides, syntheticpolynucleotides, polynucleotides, oligonucleotides, peptide nucleicacids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High MolecularWeight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA,mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA. In somecases, the nucleic acid may be genomic DNA (gDNA).

Additionally, the density of the oligonucleotide barcodes may be atleast about 1,000,000 oligonucleotide barcodes per the firstmicrocapsule. The oligonucleotide barcode may be coupled to themicrocapsule via a chemical cross-linker, such as, for example adisulfide bond.

An additional aspect of the disclosure comprises a device comprising aplurality of partitions, wherein: at least one partition of theplurality of partitions comprises a microcapsule comprising anoligonucleotide barcode; and the microcapsule is degradable upon theapplication of a stimulus to the microcapsule. The partition, forexample, may be a well or a droplet. In some cases, the microcapsulecomprises a chemical cross-linker such as, for example, a disulfidebond. Moreover, the microcapsule may comprise a polymer gel such as, forexample, a polyacrylamide gel. Also, the microcapsule may comprise abead. In some cases, the bead may be a gel bead.

The stimulus may be selected from the group consisting of a biological,chemical, thermal, electrical, magnetic, or photo stimulus, and acombination thereof. In some cases, the chemical stimulus may beselected from the group consisting of a change in pH, change in ionconcentration, and a reducing agent. The reducing agent, for example,may be dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

Furthermore, a nucleic acid may comprise the oligonucleotide barcode andthe nucleic acid may comprise a deoxyuridine triphosphate (dUTP). Insome cases, the partition may comprise a polymerase unable to accept adeoxyuridine triphosphate (dUTP). Additionally, the partition maycomprise a target analyte such as, for example, a nucleic acid. Thenucleic acid may be selected from the group consisting of DNA, RNA,dNTPs, ddNTPs, amplicons, synthetic nucleotides, syntheticpolynucleotides, polynucleotides, oligonucleotides, peptide nucleicacids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High MolecularWeight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA,mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA. In somecases, the nucleic acid may be genomic DNA (gDNA). The oligonucleotidebarcode may be coupled to the microcapsule via a chemical cross-linker.In some cases, the chemical cross-linker may be a disulfide bond.

A further aspect of the disclosure provides a method for samplepreparation comprising combining a microcapsule comprising anoligonucleotide barcode and a target analyte into a partition, whereinthe microcapsule is degradable upon the application of a stimulus to themicrocapsule; and applying the stimulus to the microcapsule to releasethe oligonucleotide barcode to the target analyte. The partition may be,for example, a well or a droplet. In some cases, the microcapsule maycomprise a polymer gel such as, for example, a polyacrylamide. Moreover,the microcapsule may comprise a bead. In some cases, the bead may be agel bead. Moreover, the microcapsule may comprise a chemicalcross-linker such as, for example, a disulfide bond.

The stimulus may be selected from the group consisting of a biological,chemical, thermal, electrical, magnetic, photo stimulus, and acombination thereof. In some cases, the chemical stimulus may beselected from the group consisting of a change in pH, change in ionconcentration, and a reducing agent. The reducing agent may be, forexample, dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

Also, a nucleic acid may comprise the oligonucleotide barcode and thenucleic acid may comprise a deoxyuridine triphosphate (dUTP). In somecases, the partition may comprise a polymerase unable to accept adeoxyuridine triphosphate (dUTP). Moreover, the method may also compriseattaching the oligonucleotide barcode to the target analyte. Theattaching may be completed, for example, with a nucleic acidamplification reaction. Moreover, the analyte may be a nucleic acid. Insome cases, the nucleic acid may be selected from the group consistingof DNA, RNA, dNTPs, ddNTPs, amplicons, synthetic nucleotides, syntheticpolynucleotides, polynucleotides, oligonucleotides, peptide nucleicacids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High MolecularWeight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA,mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA. In somecases, the nucleic acid may be genomic DNA (gDNA). Furthermore, theoligonucleotide barcode may be coupled to the microcapsule via achemical cross-linker. In some cases, the chemical cross-linker may be adisulfide bond.

A further aspect of the disclosure provides a composition comprising adegradable gel bead, wherein the gel bead comprises at least about1,000,000 oligonucleotide barcodes. In some cases, the 1,000,000oligonucleotide barcodes are identical.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entiretiesfor all purposes and to the same extent as if each individualpublication, patent, or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of a device of this disclosure are set forth withparticularity in the appended claims. A better understanding of thefeatures and advantages of this disclosure will be obtained by referenceto the following detailed description that sets forth illustrativeembodiments, in which the principles of a device of this disclosure areutilized, and the accompanying drawings of which:

FIG. 1A is a schematic representation of a microcapsule or inner reagentdroplet.

FIG. 1B is a schematic representation of a microcapsule containingmultiple inner reagent droplets.

FIG. 2A is a schematic illustration of a top down view of an exemplarymicrocapsule array.

FIG. 2B is a schematic illustration of an exemplary side view of amicrocapsule array.

FIG. 3 is a schematic illustration of a multi-microcapsule arrayconfiguration on a 96-well plate holder.

FIG. 4A is a schematic flow diagram representative of a reactionsequence in one microwell of a microwell capsule array.

FIG. 4B is similar to 4A, except that it is annotated with examples ofmethods that can be performed at each step.

DETAILED DESCRIPTION OF THE INVENTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

I. General Overview

The present disclosure provides microwell or other partition capsulearray devices and methods of using such devices. Generally, the deviceis an assembly of partitions (e.g., microwells, droplets) that areloaded with microcapsules, often at a particular concentration ofmicrocapsules per partition.

The devices may be particularly useful to perform sample preparationoperations. In some cases, a device subdivides a sample (e.g., aheterogeneous mixture of nucleic acids, a mixture of cells, etc.) intomultiple partitions such that only a portion of the sample is present ineach partition. For example, a nucleic acid sample comprising a mixtureof nucleic acids may be partitioned such that no more than one strand of(or molecule of) nucleic acid is present in each partition. In otherexamples, a cell sample may be partitioned such that no more than onecell is present in each partition.

Following the partitioning step, any of a number of different operationsmay be performed on the subdivided sample within the device. Thepartitions may include one or more capsules that contain one or morereagents (e.g., enzymes, unique identifiers (e.g., bar codes),antibodies, etc.). In some cases, the device, a companion device or auser provides a trigger that causes the microcapsules to release one ormore of the reagents into the respective partition. The release of thereagent may enable contact of the reagent with the subdivided sample.For example, if the reagent is a unique identifier such as a barcode,the sample may be tagged with the unique identifier. The tagged samplemay then be used in a downstream application such as a sequencingreaction.

A variety of different reactions and/operations may occur within adevice disclosed herein, including but not limited to: samplepartitioning, sample isolation, binding reactions, fragmentation (e.g.,prior to partitioning or following partitioning), ligation reactions,and other enzymatic reactions. The device also may be useful for avariety of different molecular biology applications including, but notlimited to, nucleic acid sequencing, protein sequencing, nucleic acidquantification, sequencing optimization, detecting gene expression,quantifying gene expression, and single-cell analysis of genomic orexpressed markers. Moreover, the device has numerous medicalapplications. For example, it may be used for the identification,detection, diagnosis, treatment, staging of, or risk prediction ofvarious genetic and non-genetic diseases and disorders including cancer.

II. Microcapsules

FIG. 1A is a schematic of an exemplary microcapsule comprising aninternal compartment 120 enveloped by a second layer 130, which isencapsulated by a solid or semi-permeable shell or membrane 110. Ingeneral, the shell separates the internal compartment(s) from theirimmediate environment (e.g., interior of a microwell). The internalcompartments, e.g., 120, 130, may comprise materials such as reagents.As depicted in FIG. 1A, the reagents 100 may be present in the internalcompartment 120. However, in some cases, the reagents are located in theenveloping layer 130 or in both compartments. Generally, themicrocapsule may release the inner materials, or a portion thereof,following the introduction of a particular trigger. The trigger maycause disruption of the shell layer 110 and/or the internal envelopinglayer 130, thereby permitting contact of the internal compartment 100,120 with the outside environment, such as the cavity of a microwell.

The microcapsule may comprise several fluidic phases and may comprise anemulsion (e.g. water-in-oil emulsion, oil-in-water emulsion). Amicrocapsule may comprise an internal layer 120 that is immiscible witha second layer 130 enveloping the internal layer. For example, theinternal layer 120 may comprise an aqueous fluid, while the envelopinglayer 130 may be a non-aqueous fluid such as an oil. Conversely, theinternal layer 120 may comprise a non-aqueous fluid (e.g., oil), and theenveloping layer 130 may comprise an aqueous fluid. In some cases, themicrocapsule does not comprise an enveloping second layer. Often, themicrocapsule is further encapsulated by a shell layer 110, which maycomprise a polymeric material. In some cases, a microcapsule maycomprise a droplet. In some cases, a microcapsule may be a droplet.

Droplets and methods for droplet generation, for example, are describedin U.S. Pat. No. RE41,780, which is incorporated herein by reference inits entirety for all purposes. The device also may contain amicrofluidic element that enables the flow of a sample and/ormicrocapsules through the device and distribution of the sample and/ormicrocapsules within the partitions.

The microcapsule can comprise multiple compartments. The microcapsulemay comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,10000, or 50000 compartments. In other cases, the microcapsule comprisesless than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000compartments. Similarly, each compartment, or a subset thereof, may alsobe subdivided into a plurality of additional compartments. In somecases, each compartment, or subset thereof, is subdivided into at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,50, 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or 50000compartments. In other cases, each compartment, or subset thereof, isfurther subdivided into less than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 50, 100, 500, 1000, 1500, 2000, 2500, 3000,3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000,9500, 10000, or 50000 compartments.

There are several possible distributions of reagent in the multiplecompartments. For example, each compartment (or some percentage of thetotal number of compartments) may comprise the same reagent or the samecombination or reagents. In some cases, each compartment (or somepercentage of the total number of compartments) comprises differentreagents or a different combination of reagents.

The compartments may be configured in a variety of ways. In some cases,the microcapsule may comprise multiple concentric compartments(repeating units of compartments that contain the precedingcompartment), often separated by an immiscible layer. In suchmicrocapsules, the reagents may be present in alternating compartments,in every third compartment, or in every fourth compartment.

In some cases, most of the compartments with a microcapsule are notconcentric; instead, they exist as separate, self-contained entitieswithin a microcapsule. FIG. 1B depicts an example of a microcapsule thatcontains a plurality of smaller microcapsules 140, each containing areagent. Like many of the other microcapsules described herein, themicrocapsule may be encapsulated by an outer shell, often comprising apolymer material 150. The plurality of smaller microcapsulesencapsulated within the larger microcapsule may be physically separatedby an immiscible fluid 160, thereby preventing mixing of reagents beforeapplication of a stimulus and release of reagents into solution. In somecases, the immiscible fluid is loaded with additional materials orreagents. In some cases, the plurality of smaller microcapsules aresurrounded by a layer of immiscible fluid (e.g., 170) which is furthersurrounded by a fluid 160 that is miscible with the inner fluid of themicrocapsules. For example, the interior microcapsules 180 may comprisean aqueous interior enveloped by an immiscible (e.g., oil) layer, thatis further surrounded by an aqueous layer 160. The miscible compartments(e.g., 160 and 180) may each contain reagents. They may contain the samereagents (or the same combination of reagents) or different reagents (ordifferent combination of reagents). Alternatively, one or some of themiscible compartments may comprise no reagents.

The microcapsule may comprise a polymeric shell (see, e.g., FIGS. 1 and2) or multiple polymeric shells. For example, the microcapsule maycomprise multiple polymeric shells layered on top of each other. Inother cases, individual compartments within a microcapsule comprise apolymeric shell, or a subset of the compartments may comprise apolymeric shell. For example, all or some of the smaller compartments140 in FIG. 1B may comprise a polymeric shell that separates them fromthe fluidic interior 160. The microcapsule may be designed so that aparticular reagent is contained within a compartment that has apolymerized shell, while a different reagent is within a compartmentthat is simply enveloped by an immiscible liquid. For example, a reagentthat is desired to be released upon a heat trigger may be containedwithin the compartments that have a heat-sensitive or heat-activatablepolymerized shell, while reagents designed to be released upon adifferent trigger may be present in different types of compartments. Inanother example, paramagnetic particles may be incorporated into thecapsule shell wall. A magnet or electric field may then be used toposition the capsule to a desired location. In some cases, a magneticfield (e.g., high frequency alternating magnetic field) can be appliedto such capsules; the incorporated paramagnetic particles may thentransform the energy of the magnetic field into heat, thereby triggeringrupture of the capsule.

The microcapsule component of a device of this disclosure may providefor the controlled and/or timed release of reagents for samplepreparation of an analyte. Microcapsules may be used in particular forcontrolled release and transport of varying types of chemicals,ingredients, pharmaceuticals, fragrances etc. . . . , includingparticularly sensitive reagents such as enzymes and proteins (see, e.g.,D. D. Lewis, “Biodegradable Polymers and Drug Delivery Systems”, M.Chasin and R. Langer, editors (Marcel Decker, New York, 1990); J. P.McGee et al., J. Control. Release 34 (1995), 77).

Microcapsules may also provide a means for delivery of reagents indiscrete and definable amounts. Microcapsules may be used to preventpremature mixing of reagents with the sample, by segregating thereagents from the sample. Microcapsules also may ease handling of—andlimit contacts with—particularly sensitive reagents such as enzymes,nucleic acids and other chemicals used in sample preparation.

A. Preparation of Microcapsules

Microcapsules of a device of this disclosure may be prepared by numerousmethods and processes. Preparative techniques may include pan coating,spray drying, centrifugal extrusion, emulsion-based methods, and/ormicrofluidic techniques. Typically, a method for preparation is chosenbased on the desired characteristics of the microcapsule. For example,shell wall thickness, permeability, chemical composition of the shellwall, mechanical integrity of the shell wall and capsule size may betaken into consideration when choosing a method. Methods of preparationmay also be selected based on the ability to incorporate specificmaterials within the capsule such as whether the core materials (e.g.,fluids, reagents, etc.) are aqueous, organic or inorganic. Additionally,preparation methods can affect the shape and size of the microcapsule.For example a capsule's shape, (e.g., spherical, ellipsoidal, etc.), maydepend on the shape of the droplet in the precursor liquid which may bedetermined by the viscosity and surface tension of the core liquid,direction of flow of the emulsion, the choice of surfactants used indroplet stabilization, as well as physical confinement such aspreparations made in a microchannel or capillary of a particular size(e.g., a size requiring distortion of the microcapsule in order for themicrocapsule to fit within the microchannel or capillary.

Microcapsules may be prepared through emulsification polymerization, aprocess in which monomer units at an aqueous/organic interface in anemulsion polymerize to form a shell. Reagents are mixed with the aqueousphase of the biphasic mixture. Vigorous shaking, or sonication of themixture, creates droplets containing reagents, which are encased by apolymeric shell.

In some cases, microcapsules may be prepared through layer-by-layerassembly, a process in which negatively and positively chargedpolyelectrolytes are deposited onto particles such as metal oxide cores.Electrostatic interactions between polyelectrolytes create a polymericshell around the core. The core can be subsequently removed via additionof acid, resulting in a semi-permeable hollow sphere which can be loadedwith various reagents.

In still further cases, microcapsules may be prepared throughcoacervation, a process in which two oppositely charged polymers inaqueous solution become entangled to form a neutralized polymer shellwall. One polymer may be contained within an oil phase, while the other,of opposite charge is contained in an aqueous phase. This aqueous phasemay contain reagents to be encapsulated. The attraction of one polymerfor another can result in the formation of coascervates. In someembodiments, gelatin and gum Arabic are components of this preparativemethod.

Microcapsules also may be prepared through internal phase separation, aprocess in which a polymer is dissolved in a solvent mixture containingvolatile and nonvolatile solvents. Droplets of the resultant solutionare suspended in an aqueous layer, which is stabilized by continualagitation and the use of surfactants. This phase may contain reagents tobe encapsulated. When the volatile solvent evaporates, the polymerscoalesce to form a shell wall. In some cases, polymers such aspolystyrene, poly(methyl methacrylate) and poly(tetrahydrofuran) areused to form shell walls.

Microcapsules also may be prepared through flow focusing methods, aprocess in which a microcapillary device is used to generate doubleemulsions containing a single internal droplet encased in a middle fluidwhich is then dispersed to an outer fluid. The inner droplet may containreagents to be encapsulated. The middle fluid becomes the shell wall,which can be formed via cross-linking reactions.

B. Microcapsule Composition

Microcapsules may comprise a variety of materials with a wide range ofchemical characteristics. Generally, the microcapsules comprisematerials with the ability to form microcapsules of a desired shape andsize and that are compatible with the reagents to be stored in themicrocapsules.

Microcapsules may comprise a wide range of different polymers includingbut not limited to: polymers, heat sensitive polymers, photosensitivepolymers, magnetic polymers, pH sensitive polymers, salt-sensitivepolymers, chemically sensitive polymers, polyelectrolytes,polysaccharides, peptides, proteins, and/or plastics. Polymers mayinclude but are not limited to materials such aspoly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS),poly(allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine)(PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle)(PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP),poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA),polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde)(PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL),poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

Often, materials for the microcapsules, particularly the shells ofmicrocapsules, may enable the microcapsule to be disrupted with anapplied stimulus. For example, a microcapsule may be prepared from heatsensitive polymers and/or may comprise one or more shells comprisingsuch heat-sensitive polymers. The heat-sensitive polymer may be stableunder conditions used for storage or loading. Upon exposure to heat, theheat-sensitive polymer components may undergo depolymerization,resulting in disruption to the integrity of the shell and release of theinner materials of the microcapsule (and/or of the inner microcapsules)to the outside environment (e.g., the interior of a microwell).Exemplary heat-sensitive polymers may include, but are not limited toNIPAAm or PNIPAM hydrogel. The microcapsules may also comprise one ormore types of oil. Exemplary oils include but are not limited tohydrocarbon oils, fluorinated oils, fluorocarbon oils, silicone oils,mineral oils, vegetable oils, and any other suitable oil.

The microcapsules may also comprise a surfactant, such as an emulsifyingsurfactant. Exemplary surfactants include, but are not limited to,cationic surfactants, non-ionic surfactants, anionic surfactants,hydrocarbon surfactants or fluorosurfactants. The surfactant mayincrease the stability of one or more components of the microcapsule,such as an inner compartment that comprises an oil.

Additionally, the microcapsules may comprise an inner material that ismiscible with materials external to the capsule. For example, the innermaterial may be an aqueous fluid and the sample within the microwell mayalso be in an aqueous fluid. In other examples, the microcapsule maycomprise powders or nanoparticles that are miscible with an aqueousfluid. For example, the microcapsule may comprise such powders ornanoparticles in an inner compartment. Upon disruption of themicrocapsule, such powders or nanoparticles are released into theexternal environment (e.g., interior of microwell) and may mix with anaqueous fluid (e.g., an aqueous sample fluid).

Additionally, the microcapsule may comprise a material that isimmiscible with the surrounding environment (e.g., interior ofmicrowell, sample fluid). In such cases, when the inner emulsion isreleased to the surrounding environment, the phase separation betweenthe inner and outer components may promote mixing, such as mixing of theinner components with the surrounding fluid. In some cases, when amicrocapsule is triggered to release its contents, a pressure or forceis also released that promotes mixing of internal and externalcomponents.

The microcapsules may also comprise a polymer within the interior of thecapsule. In some instances this polymer may be a porous polymer beadthat may entrap reagents or combinations of reagents. In otherinstances, this polymer may be a bead that has been previously swollento create a gel. Examples of polymer based gels that may be used asinner emulsions of capsules may include, but are not limited to sodiumalginate gel, or poly acrylamide gel swelled with oligonucleotide barcodes or the like.

In some cases, a microcapsule may be a gel bead comprising any of thepolymer based gels described herein. Gel bead microcapsules may begenerated, for example, by encapsulating one or more polymericprecursors into droplets. Upon exposure of the polymeric precursors toan accelerator (e.g., tetramethylethylenediamine (TEMED)), a gel beadmay be generated.

Analytes and/or reagents, such as oligonucleotide barcodes, for example,may be coupled/immobilized to the interior surface of a gel bead (e.g.,the interior accessible via diffusion of an oligonucleotide barcodeand/or materials used to generate an oligonucleotide barcode) and/or theouter surface of a gel bead or any other microcapsule described herein.Coupling/immobilization may be via any form of chemical bonding (e.g.,covalent bond, ionic bond) or physical phenomena (e.g., Van der Waalsforces, dipole-dipole interactions, etc.). In some cases,coupling/immobilization of a reagent to a gel bead or any othermicrocapsule described herein may be reversible, such as, for example,via a labile moiety (e.g., via a chemical cross-linker, includingchemical cross-linkers described herein). Upon application of astimulus, the labile moiety may be cleaved and the immobilized reagentset free. In some cases, the labile moiety is a disulfide bond. Forexample, in the case where an oligonucleotide barcode is immobilized toa gel bead via a disulfide bond, exposure of the disulfide bond to areducing agent can cleave the disulfide bond and free theoligonucleotide barcode from the bead. The labile moiety may be includedas part of a gel bead or microcapsule, as part of a chemical linker thatlinks a reagent or analyte to a gel bead or microcapsule, and/or as partof a reagent or analyte.

A gel bead or any other type of microcapsule described herein maycontain varied numbers of reagents. The density of a reagent permicrocapsule may vary depending on the particular microcapsule utilizedand the particular reagent. For example, a microcapsule or gel bead maycomprise at least about 1; 10; 100; 1,000; 10,000; 100,000; 1,000,000;5,000,000; 10,000,000, 50,000,000; 100,000,000; 500,000,000; or1,000,000,000 oligonucleotide barcodes per microcapsule or gel bead. Agel bead may comprise identical oligonucleotide barcodes or may comprisediffering oligonucleotide barcodes.

In other example, the microcapsule may comprise one or more materialsthat create a net neutral, negative or positive charge on the outershell wall of the capsule. In some instances, the charge of a capsulemay aid in preventing or promoting aggregation or clustering ofparticles, or adherence or repulsion to parts of the device.

In addition, the microcapsule may comprise one or more materials thatcause the outer shell wall of the capsule to be hydrophilic orhydrophobic. A hydrophilic material that may be used for capsule shellwalls may be poly(N-isopropylacrylamide). A hydrophobic material thatmay be used for capsule shell walls may be polystyrene. In certaininstances, a hydrophilic shell wall may aid in wicking of the capsuleinto wells comprising aqueous fluid.

C. Microcapsule Size and Shape

A microcapsule may be any of a number of sizes or shapes. In some cases,the shape of the microcapsule may be spherical, ellipsoidal,cylindrical, hexagonal or any other symmetrical or non-symmetricalshape. Any cross-section of the microcapsule may also be of anyappropriate shape, include but not limited to: circular, oblong, square,rectangular, hexagonal, or other symmetrical or non-symmetrical shape.In some cases, the microcapsule may be of a specific shape thatcomplements an opening (e.g., surface of a microwell) of the device. Forexample, the microcapsule may be spherical and the opening of amicrowell of the device may be circular.

The microcapsules may be of uniform size (e.g., all of the microcapsulesare the same size) or heterogeneous size (e.g., some of themicrocapsules are of different sizes). A dimension (e.g., diameter,cross-section, side, etc.) of a microcapsule may be at least about 0.001μm, 0.01 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200 μm,300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1 nm. In somecases, the microcapsule comprises a microwell that is at most about0.001 μm, 0.01 μm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 50 μm, 100 μm, 200μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm or 1 nm.

In some cases, microcapsules are of a size and/or shape so as to allow alimited number of microcapsules to be deposited in individual partitions(e.g., microwells, droplets) of the microcapsule array. Microcapsulesmay have a specific size and/or shape such that exactly or no more than1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 capsules fit into an individualmicrowell; in some cases, on average 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10capsules fit into an individual microwell. In still further cases, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, or 1000 capsules fit intoan individual microwell.

D. Reagents and Reagent Loading

The devices provided herein may comprise free reagents and/or reagentsencapsulated into microcapsules. The reagents may be a variety ofmolecules, chemicals, particles, and elements suitable for samplepreparation reactions of an analyte. For example, a microcapsule used ina sample preparation reaction for DNA sequencing of a target maycomprise one or more of the following reagents: enzymes, restrictionenzymes (e.g., multiple cutters), ligase, polymerase (e.g., polymerasesthat do and do not recognize dUTPs and/or uracil), fluorophores,oligonucleotide barcodes, buffers, deoxynucleotide triphosphates (dNTPs)(e.g. deoxyadenosine triphosphate (dATP), deoxycitidine triphosphate(dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate(dTTP), deoxyuridine triphosphate (dUTP)), deoxynucleotide triphosphates(ddNTPs) and the like. In another example, a microcapsule used in asample preparation reaction for single cell analysis may comprisereagents such as one or more of the following reagents: lysis buffer,detergent, fluorophores, oligonucleotide barcodes, ligase, proteases,heat activatable proteases, protease or nuclease inhibitors, buffer,enzymes, antibodies, nanoparticles, and the like.

Exemplary reagents include, but are not limited to: buffers, acidicsolution, basic solution, temperature-sensitive enzymes, pH-sensitiveenzymes, light-sensitive enzymes, metals, metal ions, magnesiumchloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionicbuffer, inhibitor, enzyme, protein, nucleic acid, antibodies,saccharides, lipid, oil, salt, ion, detergents, ionic detergents,non-ionic detergents, oligonucleotides, nucleotides, dNTPs, ddNTPs,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleicacids, circular DNA (cDNA), double-stranded DNA (dsDNA), single-strandedDNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA(gDNA), viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nRNA,short-interfering RNA (siRNA), small nuclear RNA (snRNA), smallnucleolar RNA (snoRNA), small Cajul body specific RNA, (scaRNA),microRNA, double-stranded RNA (dsRNA), ribozyme, riboswitch and viralRNA, polymerase (e.g., polymerases that do and do not recognize dUTPsand/or uracil), ligase, restriction enzymes, proteases, nucleases,protease inhibitors, nuclease inhibitors, chelating agents, reducingagents (e.g., dithiotheritol (DTT), 2-tris(2-carboxyethyl)phosphine(TCEP)), oxidizing agents, fluorophores, probes, chromophores, dyes,organics, emulsifiers, surfactants, stabilizers, polymers, water, smallmolecules, pharmaceuticals, radioactive molecules, preservatives,antibiotics, aptamers, and pharmaceutical drug compounds.

In some cases, a microcapsule comprises a set of reagents that have asimilar attribute (e.g., a set of enzymes, a set of minerals, a set ofoligonucleotides, a mixture of different bar-codes, a mixture ofidentical bar-codes). In other cases, a microcapsule comprises aheterogeneous mixture of reagents. In some cases, the heterogeneousmixture of reagents comprises all components necessary to perform areaction. In some cases, such mixture comprises all components necessaryto perform a reaction, except for 1, 2, 3, 4, 5, or more componentsnecessary to perform a reaction. In some cases, such additionalcomponents are contained within a different microcapsule or within asolution within a partition (e.g., microwell) of the device.

Reagents may be pre-loaded into the device (e.g., prior to introductionof analyte) or post-loaded into the device. They may be loaded directlyinto the device; or, in some cases, the reagents are encapsulated into amicrocapsule that is loaded into the device. In some cases, onlymicrocapsules comprising reagents are introduced. In other cases, bothfree reagents and reagents encapsulated in microcapsules are loaded intothe device, either sequentially or concurrently. In some cases, reagentsare introduced to the device either before or after a particular step.For example, a lysis buffer reagent may be introduced to the devicefollowing partitioning of a cellular sample into multiple partitions(e.g., microwells, droplets) within the device. In some cases, reagentsand/or microcapsules comprising reagents are introduced sequentiallysuch that different reactions or operations occur at different steps.The reagents (or microcapsules) may be also be loaded at stepsinterspersed with a reaction or operation step. For example,microcapsules comprising reagents for fragmenting molecules (e.g.,nucleic acids) may be loaded into the device, followed by afragmentation step, which may be followed by loading of microcapsulescomprising reagents for ligating bar-codes (or other unique identifiers,e.g., antibodies) and subsequent ligation of the bar-codes to thefragmented molecules. Additional methods of loading reagents aredescribed further herein in other sections.

E. Molecular ‘Barcodes’

It may be desirable to retain the option of identifying and trackingindividual molecules or analytes after or during sample preparation. Insome cases, one or more unique molecular identifiers, sometimes known inthe art as a ‘molecular barcodes,’ are used as sample preparationreagents. These molecules may comprise a variety of different forms suchas oligonucleotide bar codes, antibodies or antibody fragments,fluorophores, nanoparticles, and other elements or combinations thereof.Depending upon the specific application, molecular barcodes mayreversibly or irreversibly bind to the target analyte and allow foridentification and/or quantification of individual analytes afterrecovery from a device after sample preparation.

A device of this disclosure may be applicable to nucleic acidsequencing, protein detection, single molecule analysis and othermethods that require a) precise measurement of the presence and amountof a specific analyte b) multiplex reactions in which multiple analytesare pooled for analysis. A device of this disclosure may utilize themicrowells of the microwell array or other type of partition (e.g.,droplets) to physically partition target analytes. This physicalpartitioning allows for individual analytes to acquire one or moremolecular barcodes. After sample preparation, individual analytes may bepooled or combined and extracted from a device for multiplex analysis.For most applications, multiplex analysis substantially decreases thecost of analysis as well as increases through-put of the process, suchas in the case of the nucleic acid sequencing. Molecular barcodes mayallow for the identification and quantification of individual moleculeseven after pooling of a plurality of analytes. For example, with respectto nucleic acid sequencing, molecular barcodes may permit the sequencingof individual nucleic acids, even after the pooling of a plurality ofdifferent nucleic acids.

Oligonucleotide barcodes, in some cases, may be particularly useful innucleic acid sequencing. In general, an oligonucleotide barcode maycomprise a unique sequence (e.g., a barcode sequence) that gives theoligonucleotide barcode its identifying functionality. The uniquesequence may be random or non-random. Attachment of the barcode sequenceto a nucleic acid of interest may associate the barcode sequence withthe nucleic acid of interest. The barcode may then be used to identifythe nucleic acid of interest during sequencing, even when other nucleicacids of interest (e.g., comprising different barcodes) are present. Incases where a nucleic acid of interest is fragmented prior tosequencing, an attached barcode may be used to identify fragments asbelonging to the nucleic acid of interest during sequencing.

An oligonucleotide barcode may consist solely of a unique barcodesequence or may be included as part of an oligonucleotide of longersequence length. Such an oligonucleotide may be an adaptor required fora particular sequencing chemistry and/or method. For example, suchadaptors may include, in addition to an oligonucleotide barcode,immobilization sequence regions necessary to immobilize (e.g., viahybridization) the adaptor to a solid surface (e.g., solid surfaces in asequencer flow cell channel); sequence regions required for the bindingof sequencing primers; and/or a random sequence (e.g., a random N-mer)that may be useful, for example, in random amplification schemes. Anadaptor can be attached to a nucleic acid to be sequenced, for example,by amplification, ligation, or any other method described herein.

Moreover, an oligonucleotide barcode, and/or a larger oligonucleotidecomprising an oligonucleotide barcode may comprise natural nucleic acidbases and/or may comprise non-natural bases. For example, in cases wherean oligonucleotide barcode or a larger oligonucleotide comprising anoligonucleotide barcode is DNA, the oligonucleotide may comprise thenatural DNA bases adenine, guanine, cytosine, and thymine and/or maycomprise non-natural bases such as uracil.

F. Microcapsule-Preparation for Microwell Loading

Following preparation, reagent loaded microcapsules may be loaded into adevice using a variety of methods. Microcapsules, in some instances, maybe loaded as ‘dry capsules.’ After preparation, capsules may beseparated from a liquid phase using various techniques, including butnot limited to differential centrifugation, evaporation of the liquidphase, chromatography, filtration and the like. ‘Dry capsules’ may becollected as a powder or particulate matter and then deposited intomicrowells of the microwell array. Loading ‘dry capsules’ may be apreferred method in instances in which loading of ‘wet capsules,’ leadsto inefficiencies of loading such as empty wells and poor distributionof microcapsules across the microwell array.

Reagent-loaded microcapsules may also be loaded into a device when themicrocapsules are within a liquid phase, and thereby loaded as ‘wetcapsules.’ In some instances, microcapsules may be suspended in avolatile oil such that the oil can be removed or evaporated, leavingonly the dry capsule in the well. Loading ‘wet capsules’ may be apreferred method in some instances in which loading of dry capsulesleads to inefficiencies of loading, such as microcapsule clustering,aggregation and poor distribution of microcapsules across the microwellarray. Additional methods of loading reagents and microcapsules aredescribed in other sections of this disclosure.

The microcapsules also may have a particular density. In some cases, themicrocapsules are less dense than an aqueous fluid (e.g., water); insome cases, the microcapsules are denser than an aqueous fluid (e.g.,water). In some cases, the microcapsules are less dense than anon-aqueous fluid (e.g., oil); in some cases, the microcapsules aredenser than a non-aqueous fluid (e.g., oil). Microcapsules may comprisea density at least about 0.05 g/cm³, 0.1 cm³, 0.2 g/cm³, 0.3 g/cm³, 0.4g/cm³, 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³, 0.8 g/cm³, 0.81 g/cm³, 0.82g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87 g/cm³, 0.88g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93 g/cm³, 0.94g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99 g/cm³, 1.00g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4 g/cm³, 1.5g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³, 2.1 g/cm³,2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. In other cases,microcapsule densities may be at most about 0.7 g/cm³, 0.8 g/cm³, 0.81g/cm³, 0.82 g/cm³, 0.83 g/cm³, 0.84 g/cm³, 0.85 g/cm³, 0.86 g/cm³, 0.87g/cm³, 0.88 g/cm³, 0.89 g/cm³, 0.90 g/cm³, 0.91 g/cm³, 0.92 g/cm³, 0.93g/cm³, 0.94 g/cm³, 0.95 g/cm³, 0.96 g/cm³, 0.97 g/cm³, 0.98 g/cm³, 0.99g/cm³, 1.00 g/cm³, 1.05 g/cm³, 1.1 g/cm³, 1.2 g/cm³, 1.3 g/cm³, 1.4g/cm³, 1.5 g/cm³, 1.6 g/cm³, 1.7 g/cm³, 1.8 g/cm³, 1.9 g/cm³, 2.0 g/cm³,2.1 g/cm³, 2.2 g/cm³, 2.3 g/cm³, 2.4 g/cm³, or 2.5 g/cm³. Such densitiescan reflect the density of the microcapsule in any particular fluid(e.g., aqueous, water, oil, etc.)

III. Microwell Array

A. Structure/Features

A device of this disclosure may be a microwell array comprising a solidplate containing a plurality of holes, cavities or microwells in whichmicrocapsules and/or analytes are deposited. Generally, a fluidic sample(or analyte) is introduced into the device (e.g., through an inlet) andthen travels through a flow channel which distributes the sample intomultiple microwells. In some cases, additional fluid is introduced intothe device as well. The microwells may comprise microcapsules when thesample is introduced; or, in some cases, the microcapsules areintroduced into the microwells following introduction of the sample.

FIG. 2A depicts a prototype microwell array; a sideview is depicted inFIG. 2B. The microwell array may include a plate 220 that can be made ofany suitable material commonly used in a chemical laboratory, includingfused silica, soda lima glass, borosilicate glass, PMMA, sapphire,silicon, germanium, cyclic olefin copolymer and cyclic polymer,polyethylenes, polypropylenes, polyacrylates, polycarbonates, plastics,Topas, and other suitable substrates known in the art. The plate 220 mayinitially be a flat solid plate comprising a regular pattern ofmicrowells 270. The microwells may be formed by drilling or chemicaldissolution or any other suitable method of machining; however, plateswith a desired hole pattern are preferably molded, e.g. byinjection-molding, embossing, or using a suitable polymer, such ascyclic olefin copolymer.

The microwell array may comprise an inlet (200 and 240) and/or an outlet(210 and 260); in some cases, the microwell array comprises multipleinlets and/or outlets. A sample (or analyte) or microcapsules may beintroduced to the device via the inlet. Solutions containing analytes,reagents and/or microcapsules may be manually applied to the inlet port200 and 240 (or to a conduit attached to the inlet port) via a pipette.In some cases, a liquid handling device is used to introduce analytes,reagents, and/or microcapsules to the device. Exemplary liquid handlingdevices may rely on a pipetting robot, capillary action, or dipping intoa fluid. In some cases, the inlet port is connected to a reservoircomprising microcapsules or analytes. The inlet port may be attached toa flow channel 250 that permits distribution of the analyte, sample, ormicrocapsules to the microwells in the device. In some cases, the inletport may be used to introduce to the device a fluid (e.g., oil, aqueous)that does not contain microcapsules or analyte, such as a carrier fluid.The carrier fluid may be introduced via the inlet port before, during,or following the introduction of analyte and/or microcapsules. In caseswhere the device has multiple inlets, the same sample may be introducedvia the multiple inlets, or each inlet may convey a different sample. Insome cases, one inlet may convey a sample or analyte to the microwells,while a different inlet conveys free reagents and/or reagentsencapsulated in microcapsules to the device. The device may have atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 inlets and/or outlets.

In some cases, solutions containing microcapsules and/or analytes may bepulled through the device via a vacuum manifold attached to the outletport 210 and 260. Such manifold may apply a negative pressure to thedevice. In other cases, a positive pressure is used to move sample,analytes, and/or microcapsules through the device. The area, length, andwidth of surfaces of 230 according to this disclosure may be variedaccording to the requirements of the assay to be performed.Considerations may include, for example, ease of handling, limitationsof the material(s) of which the surface is formed, requirements ofdetection or processing systems, requirements of deposition systems(e.g. microfluidic systems), and the like. The thickness may comprise athickness of at least about 0.001 mm, 0.005 mm, 0.01 mm, 0.05 mm, 0.1mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0mm, 2.0 mm, 3.0 mm, 4.0 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm. In other cases, microcapsulethickness may be at most 0.001 mm, 0.005 mm, 0.01 mm, 0.05 mm, 0.1 mm,0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm,2.0 mm, 3.0 mm, 4.0 mm, 5.0 mm, 6.0 mm, 7.0 mm, 8.0 mm, 9.0 mm, 10.0 mm,11 mm, 12 mm, 13 mm, 14 mm, or 15 mm.

The microwells 270 can be any shape and size suitable for the assayperformed. The cross-section of the microwells may have across-sectional dimension that is circular, rectangular, square,hexagonal, or other symmetric or non-symmetric shape. In some cases, theshape of the microwell may be cylindrical, cubic, conical,frustoconical, hexagonal or other symmetric or non-symmetric shape. Thediameter of the microwells 270 may be determined by the size of thewells desired and the available surface area of the plate itself.Exemplary microwells comprise diameters of at least 0.01 μm, 0.1 μm, 0.2μm, 0.3 μm, 0.4 μm, 0.5 μm, 1 μm, 10 μm, 25 μm, 50 μm, 75 μm, 100 μm,200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, 1.0 mm.In other cases, microwell diameters may comprise at most 0.01 μm, 0.1μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 1 μm, 10 μm, 25 μm, 50 μm, 75 μm,100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μmor 1.0 mm.

The capacity (or volume) of each well can be a measure of the height ofthe well (the thickness of the plate) and the effective diameter of eachwell. The capacity of an individual well may be selected from a widerange of volumes. In some cases, the device may comprise a well (ormicrowell) with a capacity of at least 0.001 fL, 0.01 fL, 0.1 fL, 0.5fL, 1 fL, 5 fL, 10 fL, 50 fL, 100 fL, 200 fL, 300 fL, 400 fL, 500 fL,600 fL, 700 fL, 800 fL, 900 fL, 1 pL, 5 pL, 10 pL, 50 pL, 100 pL, 200pL, 300 pL, 400 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL, 1 nL, 5 nL,10 nL, 50 nL, 100 nL, 200 nL, 300 nL, 400 nL, 500 nL, 1 uL, 50 uL, or100 uL. In other cases, the microcapsule comprises a microwell that isless than 0.001 fL, 0.01 fL, 0.1 fL, 0.5 L, 5 fL, 10 fL, 50 fL, 100 fL,200 fL, 300 fL, 400 fL, 500 fL, 600 fL, 700 fL, 800 fL, 900 fL, 1 pL, 5pL, 10 pL, 50 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 600 pL, 700pL, 800 pL, 900 pL, 1 nL, 5 nL, 10 nL, 50 nL, 100 nL, 200 nL, 300 nL,400 nL, 500 nL, 1 uL, 50 uL, or 100 uL.

There may be variability in the volume of fluid in different microwellsin the array. More specifically, the volume of different microwells mayvary by at least (or at most) plus or minus 1%, 2%, 3%, 4%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or1000% across a set of microwells. For example, a microwell may comprisea volume of fluid that is at most 80% of the fluid volume within asecond microwell.

Based on the dimension of individual microwells and the size of theplate, the microwell array may comprise a range of well densities. Insome examples, a plurality of microwells may have a density of at leastabout 2,500 wells/cm², at least about 1,000 wells/cm². in some cases,the plurality of wells may have a density of at least 10 wells/cm². Inother cases, the well density may comprise at least 10 wells/cm², 50wells/cm², 100 wells/cm², 500 wells/cm², 1000 wells/cm², 5000 wells/cm²,10000 wells/cm², 50000 wells/cm², or 100000 wells/cm². In other cases,the well density may be less than 100000 wells/cm², 10000 wells/cm²,5000 wells/cm², 1000 wells/cm², 500 wells/cm², or 100 wells/cm².

In some cases, the interior surface of the microwells comprises ahydrophilic material that preferably accommodates an aqueous sample; insome cases, the region between the microwells is composed of ahydrophobic material that may preferentially attract a hydrophobicsealing fluid described herein.

Multiple microwell arrays, e.g., FIG. 2B may be arranged within a singledevice. FIG. 3, 300. For example, discrete microwell array slides may bearrayed in parallel on a plate holder. In some cases, at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 25, 50 or 100 microwell arrays are arrayed inparallel. In other cases, at most 100, 50, 25, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 devices are arrayed in parallel. The microwell arrays within acommon device may be manipulated simultaneously or sequentially. Forexample, arrayed devices may be loaded with samples or capsulessimultaneously or sequentially.

B. Microwell Array Fluids

The microwell array may comprise any of a number of different fluidsincluding aqueous, non-aqueous, oils, and organic solvents, such asalcohols. In some cases, the fluid is used to carry a component, e.g.,reagent, microcapsule, or analyte, to a target location such asmicrowells, output port, etc. In other cases, the fluid is used to flushthe system. In still other cases, the fluid may be used to seal themicrowells.

Any fluid or buffer that is physiologically compatible with the analytes(e.g., cells, molecules) or reagents used in the device may be used. Insome cases, the fluid is aqueous (buffered or not buffered). Forexample, a sample comprising a population of cells suspended in abuffered aqueous solution may be introduced into the microwell array,allowed to flow through the device, and distributed to the microwells.In other cases, the fluid passing through the device is nonaqueous(e.g., oil). Exemplary non-aqueous fluids include but are not limitedto: oils, non-polar solvent, hydrocarbon oil, decane (e.g., tetradecaneor hexadecane), fluorocarbon oil, fluorinated oil, silicone oil, mineraloil, or other oil.

Often, the microcapsules are suspended in a fluid that is compatiblewith the components of the shell of the microcapsule. Fluids includingbut not limited to water, alcohols, hydrocarbon oils or fluorocarbonoils are particularly useful fluids for suspending and flowingmicrocapsules through the microarray device.

C. Further Partitioning and Sealing

After the analyte, free reagents, and/or microcapsules are loaded intothe device and distributed to the microwells, a sealing fluid may beused to further partition or isolate them within the microwells. Thesealing fluid may also be used to seal the individual wells. The sealingfluid may be introduced through the same inlet port that was used tointroduce the analyte, reagents and/or microcapsules. But in some cases,the sealing fluid is introduced to the device by a separate inlet port,or through multiple separate inlet ports.

Often, the sealing fluid is a non-aqueous fluid (e.g., oil). When thesealing fluid flows through the microwell array device, it may displaceexcess aqueous solution (e.g., solution comprising analytes, freereagents and/or microcapsules) from individual microwells, therebypotentially removing aqueous bridges between adjacent microwells. Thewells themselves, as described herein, may comprise a hydrophilicmaterial that enables wicking of the aqueous fluids (e.g., sample fluid,microcapsule fluid) into individual wells. In some cases, regionsexternal to the wells comprise hydrophobic material, again to encouragethe positioning of the aqueous fluid into the interior of themicrowells.

The sealing fluid may either remain in the device or be removed. Thesealing fluid may be removed, e.g., by flowing through the outlet port.In other cases, the sealing oil may comprise a volatile oil that can beremoved by the application of heat. Once the sealing fluid is removed,analytes, free reagents and/or microcapsules may be physicallypartitioned from one another in the microwells.

A fluid may be selected such that its density is equal to, greater thanor less than the density of the microcapsules. For example, themicrocapsules may be denser than the sealing oil and/or aqueous fluid ofthe sample and reagents, thereby enabling the microcapsules to remain inthe microwells as the sealing oil flows through the device. In anotherexample, the capsules may be less dense than the aqueous fluid of thesample or the fluid that the microcapsules are suspended in, asdescribed herein, thereby facilitating movement and distribution of thecapsules across the plurality of microwells in a device.

In the case of microcapsules comprising paramagnetic material, amagnetic field may be used to load or direct the capsules into themicrowells. A magnetic field may also be used to retain suchmicrocapsules within the wells while the wells are being filled withsample, reagent, and/or sealing fluids. The magnetic field may also beused to remove capsule shells from the wells, particularly followingrupture of the capsules.

In some cases, the sealing fluid may remain in the microwells whenoperations or reactions are conducted therein. The presence of thesealing fluid may act to further partition, isolate, or seal theindividual microwells. In other cases, the sealing fluid may act as acarrier for the microcapsules. For example, sealing fluid comprisingmicrocapsules may be introduced to the device to facilitate distributionof the microcapsules to the individual microwells. For suchapplications, the sealing fluid may be denser than the microcapsules inorder to encourage more even distribution of the microcapsules to themicrowells. Upon application of a stimulus, the microcapsules within thesealing fluid may release reagents to the microwell. In some cases, thesealing fluid may comprise a chemical or other agent capable oftraveling from the sealing fluid to a well (e.g., by leaching or othermechanism) and triggering capsule rupture, where the capsule is presentwithin the microwell or within the sealing fluid.

Methods other than those involving sealing fluids may also be used toseal the microwells following the loading of the analyte, free reagents,and/or microcapsules. For example, the microwells may be sealed with alaminate, tape, plastic cover, oils, waxes, or other suitable materialto create an enclosed reaction chamber. The sealants described hereinmay protect the contents of the microwells from evaporation or otherunintended consequences of the reactions or operations. Prevention ofevaporation may be particularly necessary when heat is applied to thedevice, e.g., when heat is applied to stimulate microcapsule release.

In some cases, the laminate seal may also allow recovery of contentsfrom individual wells. In this case, a single well of interest may beunsealed (e.g., by removal of the laminate seal) at a given time inorder to enable further analysis of an analyte such as by MALDI massspectrometry. Such applications may be useful in a number of settings,including high-throughput drug screening.

III. Loading Step(s)

As described herein, analytes, free reagents, and/or microcapsules maybe loaded into the present device in any appropriate manner or order.The loading may be random or non-random. In some cases, a precise numberof analytes and/or microcapsules are loaded into each individualmicrowell. In some cases, a precise number of analytes and/ormicrocapsules are loaded into a particular subset of microwells in theplate. In still other cases, an average number of analytes and/ormicrcocapsules are loaded into each individual microwell. Furthermore,as described herein, in some cases, “dry” microcapsules are loaded intothe device, while in other cases “wet” microcapsules are loaded into thedevice. In some cases, a combination of “dry” and “wet” microcapsulesand/or reagents are loaded into the device, either simultaneously orsequentially.

As mentioned herein, the loading of the device may occur in any orderand may occur in multiple stages. In some cases, the microcapsules arepre-loaded into the device, prior to the loading of the analyte. Inother cases, the microcapsules and analyte are loaded concurrently. Instill other cases, the analytes are loaded before the microcapsules areloaded.

The microcapsules and/or analytes may be loaded in multiple stages ormultiple times. For example, microcapsules may be loaded into the deviceboth prior to and after analytes are loaded into the device. Themicrocapsules that are pre-loaded (e.g., loaded prior to the analyteintroduction) may comprise the same reagents as the microcapsules loadedafter the analyte introduction. In other cases, the pre-loadedmicrocapsules contain reagents that are different from the reagentswithin the microcapsules loaded after analyte introduction. In somecases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 different setsof microcapsules are loaded onto the device. In some cases, thedifferent sets of microcapsules are loaded sequentially; or, differentsets of microcapsules may also be loaded simultaneously. Similarly,multiple sets of analytes can be loaded into the device. In some cases,at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 different sets ofanalytes are loaded onto the device. In some cases, the different setsof analytes are loaded sequentially; or, different sets of analytes mayalso be loaded simultaneously.

This disclosure provides devices comprising certain numbers ofmicrocapsules and/or analytes loaded per well. In some cases, at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 100 microcapsulesand/or analytes are loaded into each individual microwell. In somecases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75,or 100 microcapsules and/or analytes are loaded into each individualmicrowell. In some cases, on average, at most 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 75, or 100 microcapsules and/or analytes areloaded into each individual microwell. In other cases, on average, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, or 100microcapsules and/or analytes are loaded into each individual microwell.In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,75, or 100 microcapsules and/or analytes are loaded into each individualmicrowell.

Analytes and/or microcapsules may be applied in a quantity that allows adesired number of analytes to be deposited into an individual microwell.For example, terminal dilution of analytes, such as cells, may achievethe loading of one cell per one microwell or any desired number ofanalytes per microwell. In some cases, a Poisson distribution is used todirect or predict the final concentration of analytes or microcapsulesper well.

The microcapsules may be loaded into the microarray device in aparticular pattern. For example, certain sections of the device maycomprise microcapsules containing a particular reagent (e.g., uniquebar-code, enzyme, antibody, antibody subclass, etc.), while othersections of the device may comprise microcapsules containing a differentreagent (e.g., a different bar-code, different enzyme, differentantibody different antibody subclass, etc.). In some cases, themicrocapsules in one section of the array may contain control reagents.For example, they may contain positive controls that include a controlanalyte and necessary materials for a reaction. Or, in some cases, themicrocapsules contain negative control reagents such as deactivatedenzyme, or a synthetic oligonucleotide sequence that is resistant tofragmentation. In some cases, negative control reagents may control forthe specificity of the sample preparation reaction etc. In other cases,the negative control microcapsules may comprise the same reagentspresent in other microcapsules except that the negative controlmicrocapsule may lack a certain reagent (e.g., lysis buffer, polymerase,etc.).

The analytes/sample also may be loaded into the microarray device in aparticular pattern. For example, certain sections of the device maycomprise particular analytes, such as control analytes or analytesderiving from a particular source. This may be used in combination withspecific loading of bar codes into known well locations. This featuremay allow mapping of specific locations on the array to sequence data,thereby reducing the number of bar codes to be used for labelingreactions.

In cases where a partition is a droplet, an analyte and reagents may becombined within the droplet with the aid of a microfluidic device. Forexample, a droplet may be generated that comprises a gel bead (e.g.,comprising an oligonucleotide barcode) a nucleic acid analyte, and anyother desired reagents. The gel bead, nucleic acid analyte, and reagentsin an aqueous phase may be combined at a junction of two or morechannels of a microfluidic device. At a second junction of two or morechannels of the microfluidic device, a droplet comprising the resultingmixture may be generated by contacting the aqueous mixture of reagents,gel bead, and nucleic acid analyte with an oil continuous phase.

IV. Microcapsule Stimuli

Various different stimuli may be used to trigger release of reagentsfrom the microcapsules, or from internal compartments therein. In somecases, a microcapsule is degradable. Generally, the trigger may causedisruption or degradation of the shell or membrane enveloping themicrocapsule, disruption or degradation of the interior of amicrocapsule, and/or disruption or degradation of any chemical bondsthat immobilize a reagent to the microcapsule. Exemplary triggersinclude but are not limited to: chemical triggers, bulk changes,biological triggers, light triggers, thermal triggers, magnetictriggers, and any combination thereof. See, e.g., Esser-Kahn et al.,(2011) Macromolecules 44: 5539-5553; Wang et al., (2009) ChemPhysChem10:2405-2409;

A. Chemical Stimuli and Bulk Changes

Numerous chemical triggers may be used to trigger the disruption ordegradation of the microcapsules. Examples of these chemical changes mayinclude, but are not limited to pH-mediated changes to the shell wall,disintegration of the shell wall via chemical cleavage of crosslinkbonds, triggered depolymerization of the shell wall, and shell wallswitching reactions. Bulk changes may also be used to trigger disruptionof the microcapsules.

A change in pH of the solution, particularly a decrease in pH, maytrigger disruption via a number of different mechanisms. The addition ofacid may cause degradation or disassembly of the shell wall through avariety of mechanisms. Addition of protons may disassemble cross-linkingof polymers in the shell wall, disrupt ionic or hydrogen bonds in theshell wall, or create nanopores in the shell wall to allow the innercontents to leak through to the exterior. In some examples, themicrocapsule comprises acid-degradable chemical cross-linkers such aketals. A decrease in pH, particular to a pH lower than 5, may inducethe ketal to convert to a ketone and two alcohols and facilitatedisruption of the microcapsule. In other examples, the microcapsules maycomprise one or more polyelectrolytes (e.g., PAA, PAAm, PSS, etc.) thatare pH sensitive. A decrease in pH may disrupt the ionic- orhydrogen-bonding interactions of such microcapsules, or create nanoporestherein. In some cases, microcapsules comprising polyelectrolytescomprise a charged, gel-based core that expands and contracts upon achange of pH.

Removal of cross-linkers (e.g., disulfide bonds) within themicrocapsules can also be accomplished through a number of mechanisms.In some examples, various chemicals can be added to a solution ofmicrocapsules that induce either oxidation, reduction or other chemicalchanges to polymer components of the shell wall. In some cases, areducing agent, such as beta-mercaptoethanol, dithiotheritol (DTT), or2-tris(2-carboxyethyl)phosphine (TCEP), is added such that disulfidebonds in a microcapsule shell wall are disrupted. In addition, enzymesmay be added to cleave peptide bonds within the microcapsules, therebyresulting in cleavage of shell wall cross linkers.

Depolymerization can also be used to disrupt the microcapsules. Achemical trigger may be added to facilitate the removal of a protectinghead group. For example, the trigger may cause removal of a head groupof a carbonate ester or carbamate within a polymer, which in turn causesdepolymerization and release of reagents from the inside of the capsule.

Shell wall switching reactions may be due to any structural change tothe porosity of the shell wall. The porosity of a shell wall may bemodified, for example, by the addition of azo dyes or viologenderivatives. Addition of energy (e.g., electricity, light) may also beused to stimulate a change in porosity.

In yet another example, a chemical trigger may comprise an osmotictrigger, whereby a change in ion or solute concentration of microcapsulesolution induces swelling of the capsule. Swelling may cause a buildupof internal pressure such that the capsule ruptures to release itscontents.

It is also known in the art that bulk or physical changes to themicrocapsule through various stimuli also offer many advantages indesigning capsules to release reagents. Bulk or physical changes occuron a macroscopic scale, in which capsule rupture is the result ofmechano-physical forces induced by a stimulus. These processes mayinclude, but are not limited to pressure induced rupture, shell wallmelting, or changes in the porosity of the shell wall.

B. Biological Stimuli

Biological stimuli may also be used to trigger disruption or degradationof microcapsules. Generally, biological triggers resemble chemicaltriggers, but many examples use biomolecules, or molecules commonlyfound in living systems such as enzymes, peptides, saccharides, fattyacids, nucleic acids and the like. For example, microcapsules maycomprise polymers with peptide cross-links that are sensitive tocleavage by specific proteases. More specifically, one example maycomprise a microcapsule comprising GFLGK peptide cross links. Uponaddition of a biological trigger such as the protease Cathepsin B, thepeptide cross links of the shell well are cleaved and the contents ofthe capsule are released. In other cases, the proteases may beheat-activated. In another example, microcapsules comprise a shell wallcomprising cellulose. Addition of the hydrolytic enzyme chitosan servesas biologic trigger for cleavage of cellulosic bonds, depolymerizationof the shell wall, and release of its inner contents.

C. Thermal Stimuli

The microcapsules may also be induced to release their contents upon theapplication of a thermal stimulus. A change in temperature can cause avariety changes to the microcapsule. A change in heat may cause meltingof a microcapsule such that the shell wall disintegrates. In othercases, the heat may increase the internal pressure of the innercomponents of the capsule such that the capsule ruptures or explodes. Instill other cases, the heat may transform the capsule into a shrunkendehydrated state. The heat may also act upon heat-sensitive polymerswithin the shell of a microcapsule to cause disruption of themicrocapsule.

In one example, a microcapsule comprises a thermo-sensitive hydrogelshell encapsulating one or more emulsified reagent particles. Upon theapplication of heat, such as above 35 C, the hydrogel material of theouter shell wall shrinks. The sudden shrinkage of the shell ruptures thecapsule and allows the reagents of the inside of the capsule to squirtout in the sample preparation solution in the microwell.

In some cases, the shell wall may comprise a diblock polymer, or amixture of two polymers, with different heat sensitivities. One polymermay be particularly likely to shrink after the application of heat,while the other is more heat-stable. When heat is applied to such shellwall, the heat-sensitive polymer may shrink, while the other remainsintact, causing a pore to form. In still other cases, a shell wall maycomprise magnetic nanoparticles. Exposure to a magnetic field may causethe generation of heat, leading to rupture of the microcapsule.

D. Magnetic Stimuli

Inclusion of magnetic nanoparticles to the shell wall of microcapsulesmay allow triggered rupture of the capsules as well as guide theparticles in an array. A device of this disclosure may comprise magneticparticles for either purpose. In one example, incorporation of Fe3O4nanoparticles into polyelectrolyte containing capsules triggers rupturein the presence of an oscillating magnetic field stimulus.

E. Electrical and Light Stimuli

A microcapsule may also be disrupted or degraded as the result ofelectrical stimulation. Similar to magnetic particles described in theprevious section, electrically sensitive particles can allow for bothtriggered rupture of the capsules as well as other functions such asalignment in an electric field, electrical conductivity or redoxreactions. In one example, microcapsules containing electricallysensitive material are aligned in an electric field such that release ofinner reagents can be controlled. In other examples, electrical fieldsmay induce redox reactions within the shell wall itself that mayincrease porosity.

A light stimulus may also be used to disrupt the microcapsules. Numerouslight triggers are possible and may include systems that use variousmolecules such as nanoparticles and chromophores capable of absorbingphotons of specific ranges of wavelengths. For example, metal oxidecoatings can be used as capsule triggers. UV irradiation ofpolyelectrolyte capsules coated with SiO2/TiO2 may result indisintegration of the capsule wall. In yet another example, photoswitchable materials such as azobenzene groups may be incorporated inthe shell wall. Upon the application of UV or visible light, chemicalssuch as these undergo a reversible cis-to-trans isomerization uponabsorption of photons. In this aspect, incorporation of photo switchesresult in a shell wall that may disintegrate or become more porous uponthe application of a light trigger.

F. Application of Stimuli

A device of this disclosure may be used in combination with anyapparatus or device that provides such trigger or stimulus. For example,if the stimulus is thermal, a device may be used in combination with aheated or thermally controlled plate, which allows heating of themicrowells and may induce the rupture of capsules. Any of a number ofheat transfers may be used for thermal stimuli, including but notlimited to applying heat by radiative heat transfer, convective heattransfer, or conductive heat transfer. In other cases, if the stimulusis a biological enzyme, the enzyme may be injected into a device suchthat it is deposited into each microwell. In another aspect, if thestimulus is a magnetic or electric field, a device may be used incombination with a magnetic or electric plate.

A chemical stimulus may be added to a partition and may exert itsfunction at various times after contacting a chemical stimulus with amicrocapsule. The speed at which a chemical stimulus exerts its effectmay vary depending on, for example, the amount/concentration of achemical stimulus contacted with a microcapsule and/or the particularchemical stimulus used. For example, a droplet may comprise a degradablegel bead (e.g., a gel bead comprising chemical cross-linkers, such as,for example, disulfide bonds). Upon droplet formation, a chemicalstimulus (e.g., a reducing agent) may be included in the droplet withthe gel bead. The chemical stimulus may degrade the gel bead immediatelyon contact with the gel bead, soon after (e.g., about 0, 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 min) contact with the gel bead, or at a latertime. In some cases, degradation of the gel bead may occur before,during, or after a further processing step, such as, for example, athermal cycling step as described herein.

V. Sample Preparation, Reaction and Recovery

After application of the stimulus, rupturing of capsules and release ofthe reagents, the sample preparation reaction may proceed in a device.Reactions within a device may be incubated for various periods of timesdepending on the reagents used in the sample reactions. A device mayalso be used in combination with other devices that aid in the samplepreparation reaction. For example, if PCR amplification is desired, adevice may be used in combination with a PCR thermocycler. In somecases, a thermocycler may comprise a plurality of wells. In cases wherepartitions are droplets, the droplets may be entered into the wells ofthe thermocycler. In some cases, each well may comprise multipledroplets, such that when thermal cycling is initiated, multiple dropletsare thermal cycled in each well. In another example, if the reactionrequires agitation, a device may be used in combination with a shakingapparatus.

Following the completion of the sample preparation reaction, theanalytes and products of the sample reactions may be recovered. In somecases, a device may utilize a method comprising the application ofliquid or gas to flush out the contents of the individual microwells. Inone example, the liquid comprises an immiscible carrier fluid thatpreferentially wets the microwell array material. It may also beimmiscible with water so as to flush the reaction products out of thewell. In another example, the liquid may be an aqueous fluid that can beused to flush out the samples out of the wells. After flushing of thecontents of the microwells, the contents of the microwells are pooledfor a variety of downstream analyses and applications.

VI. Applications

FIG. 4A provides a general flow of many of the methods of the presentdisclosure; and FIG. 4B provides a generally annotated version of 4A.One or more microcapsule(s) that contain reagents 410 may be pre-loadedinto microwells, followed by addition of an analyte, which, in thisparticular Figure, is a nucleic acid analyte 420. The microwells maythen be sealed 430 by any method, such as by application of a sealingfluid. The inlet and outlet ports may also be sealed, for example toprevent evaporation. Following these steps, a stimulus (e.g., heat,chemical, biological, etc.) may be applied to the microwells in order todisrupt the microcapsules 460 and trigger release of the reagents 450 tothe interior of the microwell. Subsequently, an incubation step 440 mayoccur in order to enable the reagents perform a particular function suchas lysis of cells, digestion of protein, fragmentation of high molecularweight nucleic acids, or ligation of oligonucleotide bar codes.Following the incubation step (which is optional), the contents of themicrowells may be recovered either singly or in bulk.

A. Analytes

A device of this disclosure may have a wide variety of uses in themanipulation, preparation, identification and/or quantification ofanalytes. In some cases, the analyte is a cell or population of cells.The population of cells may be homogeneous (e.g., from a cell line, ofthe same cell type, from the same type of tissue, from the same organ,etc.) or heterogenous (mixture of different types of cells). The cellsmay be primary cells, cell lines, recombinant cells, primary cells,encapsulated cells, free cells, etc.

The analytes may also be molecules, including but not limited to:polypeptides, proteins, antibodies, enzymes, nucleic acids, saccharides,small molecules, drugs, and the like. Examples of nucleic acids includebut are not limited to: DNA, RNA, dNTPs, ddNTPs, amplicons, syntheticnucleotides, synthetic polynucleotides, polynucleotides,oligonucleotides, peptide nucleic acids, cDNA, dsDNA, ssDNA, plasmidDNA, cosmid DNA, high Molecular Weight (MW) DNA, chromosomal DNA,genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA,rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA,ribozyme, riboswitch and viral RNA (e.g., retroviral RNA).

In some cases, the analytes are pre-mixed with one or more additionalmaterials, such as one or more reagents (e.g., ligase, protease,polymerase) prior to being loaded into the device. In some cases, theanalytes are pre-mixed with microcapsules comprising one or morereagents prior to being loaded onto the device.

The samples may be derived from a variety of sources including human,mammal, non-human mammal, ape, monkey, chimpanzee, plant, reptilian,amphibian, avian, fungal, viral or bacterial sources. Samples such ascells, nucleic acids and proteins may also be obtained from a variety ofclinical sources such as biopsies, aspirates, blood draws, urinesamples, formalin fixed embedded tissues and the like.

A device of this disclosure may also enable the analytes to be tagged ortracked in order to permit subsequent identification of an origin of theanalytes. This feature is in contrast with other methods that use pooledor multiplex reactions and that only provide measurements or analyses asan average of multiple samples. Here, the physical partitioning andassignment of a unique identifier to individual analytes allowsacquisition of data from individual samples and is not limited toaverages of samples.

In some examples, nucleic acids or other molecules derived from a singlecell may share a common tag or identifier and therefore may be lateridentified as being derived from that cell. Similarly, all of thefragments from a single strand of nucleic acid may be tagged with thesame identifier or tag, thereby permitting subsequent identification offragments with similar phasing or linkage on the same strand. In othercases, gene expression products (e.g., mRNA, protein) from an individualcell may be tagged in order to quantify expression. In still othercases, the device can be used as a PCR amplification control. In suchcases, multiple amplification products from a PCR reaction can be taggedwith the same tag or identifier. If the products are later sequenced anddemonstrate sequence differences, differences among products with thesame identifier can then be attributed to PCR error.

The analytes may be loaded onto the device before, after, or duringloading of the microcapsules and/or free reagents. In some cases, theanalytes are encapsulated into microcapsules before loading into themicrocapsule array. For example, nucleic acid analytes may beencapsulated into a microcapsule, which is then loaded onto the deviceand later triggered to release the analytes into an appropriatemicrowell.

Any analytes, such as DNA or cells, may be loaded in solution or asanalytes encapsulated in a capsule. In some cases, homogeneous orheterogeneous populations of molecules (e.g., nucleic acids, proteins,etc.) are encapsulated into microcapsules and loaded onto the device. Insome cases, homogeneous or heterogeneous populations of cells areencapsulated into microcapsules and loaded onto the device. Themicrocapsules may comprise a random or specified number of cells and/ormolecules. For example, the microcapsules may comprise no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500,1000, 5000, or 10000 cells and/or molecules per microcapsule. In otherexamples, the microcapsules comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 5000, or 10000 cellsand/or molecules per microcapsule. Fluidic techniques and any othertechniques may be used to encapsulate the cells and/or molecules intothe microcapsules.

Generally, the methods and compositions provided herein are useful forpreparation of an analyte prior to a down-stream application such as asequencing reaction. Often, a sequencing method is classic Sangersequencing. Sequencing methods may include, but are not limited to:high-throughput sequencing, pyrosequencing, sequencing-by-synthesis,single-molecule sequencing, nanopore sequencing, sequencing-by-ligation,sequencing-by-hybridization, RNA-Seq (Illumina), Digital Gene Expression(Helicos), Next generation sequencing, Single Molecule Sequencing bySynthesis (SMSS)(Helicos), massively-parallel sequencing, Clonal SingleMolecule Array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing,primer walking, and any other sequencing methods known in the art.

There are numerous examples of applications that may be conductedinstead of, or in conjunction with, a sequencing reaction, including butnot limited to: biochemical analyses, proteomics, immunoassays,profiling/fingerprinting of specific cell types, pharmaceuticalscreening, bait-capture experiments, protein-protein interaction screensand the like.

B. Assignment of Unique Identifiers to Analytes

The devices disclosed herein may be used in applications that involvethe assignment of unique identifiers, or molecular bar codes, toanalytes. Often, the unique identifier is a bar-code oligonucleotidethat is used to tag the analytes; but, in some cases, different uniqueidentifiers are used. For example, in some cases, the unique identifieris an antibody, in which case the attachment may comprise a bindingreaction between the antibody and the analyte (e.g., antibody and cell,antibody and protein, antibody and nucleic acid). In other cases, theunique identifier is a dye, in which case the attachment may compriseintercalation of the dye into the analyte molecule (such asintercalation into DNA or RNA) or binding to a probe labeled with thedye. In still other cases, the unique identifier may be a nucleic acidprobe, in which case the attachment to the analyte may comprise ahybridization reaction between the nucleic acid and the analyte. In somecases, the reaction may comprise a chemical linkage between theidentifier and the analyte. In other cases, the reaction may compriseaddition of a metal isotope, either directly to the analyte or by aprobe labeled with the isotope.

Often, the method comprises attaching oligonucleotide bar codes tonucleic acid analytes through an enzymatic reaction such as a ligationreaction. For example, the ligase enzyme may covalently attach a DNA barcode to fragmented DNA (e.g., high molecular-weight DNA). Following theattachment of the bar-codes, the molecules may be subjected to asequencing reaction.

However, other reactions may be used as well. For example,oligonucleotide primers containing bar code sequences may be used inamplification reactions (e.g., PCR, qPCR, reverse-transcriptase PCR,digital PCR, etc.) of the DNA template analytes, thereby producingtagged analytes. After assignment of bar codes to individual analytes,the contents of individual microwells may be recovered via the outletport in the device for further analyses.

The unique identifiers (e.g., oligonucleotide bar-codes, antibodies,probes, etc.) may be introduced to the device randomly or nonrandomly.In some cases, they are introduced at an expected ratio of uniqueidentifiers to microwells. For example, the unique identifiers may beloaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,100, 500, 1000, 5000, 10000, or 200000 unique identifiers are loaded permicrowell. In some cases, the unique identifiers may be loaded so thatless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000,5000, 10000, or 200000 unique identifiers are loaded per microwell. Insome cases, the average number of unique identifiers loaded permicrowell is less than, or greater than, about 0.0001, 0.001, 0.01, 0.1,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, or200000 unique identifiers per microwell.

The unique identifiers also may be loaded so that a set of one or moreidentical identifiers are introduced to a particular well. Such sets mayalso be loaded so that each microwell contains a different set ofidentifiers. For example, a population of microcapsules may be preparedsuch that a first microcapsule in the population comprises multiplecopies of identical unique identifiers (e.g., nucleic acid bar codes,etc.) and a second microcapsule in the population comprises multiplecopies of a unique identifier that differs from within the firstmicrocapsule. In some cases, the population of microcapsules maycomprise multiple microcapsules (e.g., greater than 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000, 5000, 10000,100000, 1000000, 10000000, 100000000, or 1000000000 microcapsules), eachcontaining multiple copies of a unique identifier that differs from thatcontained in the other microcapsules. In some cases, the population maycomprise greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 100, 500, 1000, 5000, 10000, 100000, 1000000, 10000000,100000000, or 1000000000 microcapsules with identical sets of uniqueidentifiers. In some cases, the population may comprise greater than 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 500, 1000,5000, 10000, 100000, 1000000, 10000000, 100000000, or 1000000000microcapsules, wherein the microcapsules each comprise a differentcombination of unique identifiers. For example, in some cases thedifferent combinations overlap, such that a first microcapsule maycomprise, e.g., unique identifiers A, B, and C, while a secondmicrocapsule may comprise unique identifiers A, B, and D. In anotherexample, the different combinations do not overlap, such that a firstmicrocapsule may comprise, e.g., unique identifiers A, B, and C, while asecond microcapsule may comprise unique identifiers D, E, and F.

The unique identifiers may be loaded into the device at an expected orpredicted ratio of unique identifiers per analyte (e.g., strand ofnucleic acid, fragment of nucleic acid, protein, cell, etc.) In somecases, the unique identifiers are loaded in the microwells so that morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000,10000, or 200000 unique identifiers are loaded per individual analyte inthe microwell. In some cases, the unique identifiers are loaded in themicrowells so that less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,50, 100, 500, 1000, 5000, 10000, or 200000 unique identifiers are loadedper individual analyte in the microwell. In some cases, the averagenumber of unique identifiers loaded per analyte is less than, or greaterthan, about 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,50, 100, 500, 1000, 5000, 10000, or 200000 unique identifiers peranalyte. When more than one identifier is present per analyte, suchidentifiers may be copies of the same identifier, or multiple differentidentifiers. For example, the attachment process may be designed toattach multiple identical identifiers to a single analyte, or multipledifferent identifiers to the analyte.

The unique identifiers may be used to tag a wide range of analytes,including cells or molecules. For example, unique identifiers (e.g., barcode oligonucleotides) may be attached to whole strands of nucleic acidsor to fragments of nucleic acids (e.g., fragmented genomic DNA,fragmented RNA). The unique identifiers (e.g., antibodies,oligonucleotides) may also bind to cells, include the external surfaceof a cell, a marker expressed on the cell or components within the cellsuch as organelles, gene expression products, genomic DNA, mitochondrialDNA, RNA, mRNA, or proteins. The unique identifiers also may be designedto bind or hybridize nucleic acids (e.g., DNA, RNA) present inpermeabilized cells, which may or may not be otherwise intact.

The unique identifiers may be loaded onto the device either singly or incombination with other elements (e.g., reagents, analytes). In somecases, free unique identifiers are pooled with the analytes and themixture is loaded into the device. In some cases, unique identifiersencapsulated in microcapsules are pooled with the analytes, prior toloading of the mixture onto the device. In still other cases, freeunique identifiers are loaded into the microwells prior to, during(e.g., by separate inlet port), or following the loading of theanalytes. In still other cases, unique identifiers encapsulated inmicrocapsules are loaded into the microwells prior to, concurrently with(e.g., by separate inlet port), or after loading of the analytes.

In many applications, it may be important to determine whetherindividual analytes each receive a different unique identifier (e.g.,oligonucleotide bar code). If the population of unique identifiersintroduced into the device is not significantly diverse, differentanalytes may possibly be tagged with identical identifiers. The devicesdisclosed herein may enable detection of analytes tagged with the sameidentifier. In some cases, a reference analyte may be included with thepopulation of analytes introduced into the device. The reference analytemay be, for example, a nucleic acid with a known sequence and a knownquantity. After the population of analytes is loaded and partitioned inthe device, unique identifiers may be attached to the analytes, asdescribed herein. If the unique identifiers are oligonucleotide barcodes and the analytes are nucleic acids, the tagged analytes maysubsequently be sequenced and quantified. These methods may indicate ifone or more fragments and/or analytes may have been assigned anidentical bar code.

A method disclosed herein may comprise loading the device with thereagents necessary for the assignment of bar codes to the analytes. Inthe case of ligation reactions, reagents including, but not limited to,ligase enzyme, buffer, adapter oligonucleotides, a plurality of uniqueidentifier DNA bar codes and the like may be loaded into the device. Inthe case of enrichment, reagents including but not limited to aplurality of PCR primers, oligonucleotides containing unique identifyingsequence, or bar code sequence, DNA polymerase, DNTPs, and buffer andthe like may be loaded into the device. The reagents may be loaded asfree reagents or as reagents encapsulated in microcapsules.

C. Nucleic Acid Sequencing

Nucleic acid sequencing may begin with the physical partitioning ofsample analytes into microwells at a particular density (e.g., about 1analyte per microwell or other density described herein). When nucleicacid bar codes are assigned to individual analytes, it may then bepossible to track individual molecules during subsequent steps such assubsequent amplification and/or sequencing steps, even if the analytesare later pooled together and treated en masse.

a. Nucleic Acid Phasing

The devices provided herein may be used to prepare analytes (e.g.,nucleic acid analytes) in such a manner that enables phasing or linkageinformation to be subsequently obtained. Such information may allow forthe detection of linked genetic variations in sequences, includinggenetic variations (e.g., SNPs, mutations, indels, copy numbervariations, transversions, translocations, inversions, etc.) that areseparated by long stretches of nucleic acids. These variations may existin either a cis or trans relationship. In cis relationships, two or moregenetic variations may exist in the same polynucleic acid molecule orstrand. In trans relationships, two or more genetic variations may existon multiple nucleic acid molecules or strands.

A method of determining nucleic acid phasing may comprise loading anucleic acid sample (e.g., a nucleic acid sample that spans a givenlocus or loci) into a device disclosed herein, distributing the samplesuch that at most one molecule of nucleic acid is present per microwell,and fragmenting the sample within the microwells. The method may furthercomprise attaching unique identifiers (e.g., bar codes) to thefragmented nucleic acids as described herein, recovering the nucleicacids in bulk, and performing a subsequent sequencing reaction on thesamples in order to detect genetic variations, such as two differentgenetic variations. The detection of genetic variations tagged with twodifferent bar codes may indicate that the two genetic variations arederived from two separate strands of DNA, reflecting a transrelationship. Conversely, the detection of two different geneticvariations tagged with the same bar codes may indicate that the twogenetic variations are from the same strand of DNA, reflecting a cisrelationship.

Phase information may be important for the characterization of theanalyte, particularly if the analyte derives from a subject at risk of,having, or suspected of a having a particular disease or disorder (e.g.,hereditary recessive disease such as Cystic Fibrosis, cancer, etc.). Theinformation may be able to distinguish between the followingpossibilities: (1) two genetic variations within the same gene on thesame strand of DNA and (2) two genetic variations within the same genebut located on separate strands of DNA. Possibility (1) may indicatethat one copy of the gene is normal and the individual is free of thedisease, while possibility (2) may indicate that the individual has orwill develop the disease, particularly if the two genetic variations aredamaging to the function of the gene when present within the same genecopy. Similarly, the phasing information may also be able to distinguishbetween the following possibilities: (1) two genetic variations, eachwithin a different gene on the same strand of DNA and (2) two geneticvariations, each within a different gene but located on separate strandsof DNA.

b. Cell-Specific Information

The devices provided herein may be used to prepare cellular analytes insuch a manner that enables cell-specific information to be subsequentlyobtained. Such information may enable detection of genetic variations(e.g., SNPs, mutations, indels, copy number variations, transversions,translocations, inversions, etc.) on a cell-by-cell basis, therebyenabling a determination of whether the genetic variation(s) are presentin the same cell or two different cells.

A method of determining nucleic acid cell-specific information maycomprise loading a cellular sample (e.g., a cellular sample from asubject) into a device disclosed herein, distributing the sample suchthat at most one cell is present per microwell, lysing the cells, andthen tagging the nucleic acids within the cells with unique identifiersusing a method described herein. In some cases, microcapsules comprisingunique identifiers are loaded in the microwell array device (eitherbefore, during, or after the loading of the cellular analytes) in such amanner that each cell is contacted with a different microcapsule. Theresulting tagged nucleic acids can then be pooled, sequenced, and usedto trace the origin of the nucleic acids. Nucleic acids with identicalunique identifiers may be determined to originate from the same cell,while nucleic acids with different unique identifiers may be determinedto originate from different cells.

In a more specific example, the methods herein may be used to detect thedistribution of oncogenic mutations across a population of cancer tumorcells. In this example, some of the cells may have a mutation, oramplification, of an oncogene (e.g., HER2, BRAF, EGFR, KRAS) on twostrands of DNA (homozygous), while others may be heterozygous for themutation, while still other cells may be wild-type and comprise nomutations or other variation in the oncogene. The methods describedherein may be able to detect these differences, and also may enablequantification of the relative numbers of homozygous, heterozygous, andwild-type cells. Such information may be used to stage a particularcancer or to monitor the progression of the cancer over time.

In some examples, this disclosure provides methods of identifyingmutations in two different oncogenes (e.g., KRAS and EGFR). If the samecell comprises genes with both mutations, this may indicate a moreaggressive form of cancer. In contrast, if the mutations are located intwo different cells, this may indicate that the cancer is more benign,or less advanced.

The following is another specific example of cell-specific sequencedetermination. In this example, a plurality of cells, such as from atumor biopsy, is loaded into a device. Single cells from the sample aredeposited into individual wells and labeled with a DNA bar code.

Loading of cells into a device may be achieved through non-randomloading. Parameters for non-random loading of analytes, such as cells,may be understood using an interference function such that:

${{\,^{``}{fraction}}\mspace{14mu} {multi}\text{-}{occupancy}^{''}} = {1 - \left\lbrack {\left( {1 - \frac{1}{N}} \right) + \frac{p}{N}} \right\rbrack^{C}}$

where

P=probability that a particular cell will attempt but not fit in thewell (measure of interference)

N=number of wells

L=number of labels=barcodes

C=number of cells

As part of sample preparation reactions, cells may be lysed and manysubsequent reactions are possible, including RNA amplification, DNAamplification or antibody screening for different target proteins andgenes in individual cells. After the reaction, the contents of the cellsmay be pooled together and could be further analyzed, such as by DNAsequencing. With each cell assigned a unique barcode, further analysesmay be possible including but not limited to quantification of differentgene levels or nucleic acid sequencing of individual cells. In thisexample, it may be determined whether the tumor comprises cells withdifferent genetic backgrounds (e.g., cancer clones and subclones). Therelative number of each type of cell may also be calculated.

c. Amplification Control

As disclosed herein, the device can be used for purposes of controllingfor amplification errors, such as PCR errors. For example, a nucleicacid sample may be partitioned into the microwells of the device.Following partitioning, the sample may be subjected to a PCRamplification reaction within the microwells. The PCR products within amicrowell may be tagged with the same unique identifier, using a methoddescribed herein. If the products are later sequenced and demonstratesequence differences, differences among products with the sameidentifier can then be attributed to PCR error.

d. Gene-expression Products Analysis

In other applications, a device may be used to detect gene product(e.g., protein, mRNA) expression levels in a sample, often on acell-by-cell basis. A sample may comprise individual cells, a pool ofmRNA extract from cells, or other collection of gene products. In someinstances, single cells may be loaded into microwells. In otherinstances, a pool of mRNA or other gene product may be loaded such thata desired quantity of mRNA molecules is loaded into individualmicrowells.

The methods provided herein may be particularly useful for RNA analysis.For example, using the methods provided herein, unique identifiers maybe assigned to mRNA analytes either directly or to cDNA products of areverse transcription reaction performed on the mRNA analytes. Thereverse transcription reaction may be conducted within the microwells ofthe device following loading of the analytes. Reagents for the reactionmay include but are not limited to reverse transcriptase, DNA polymeraseenzyme, buffer, dNTPs, oligonucleotide primers, oligonucleotide primerscontaining bar code sequences and the like. One or more reagents may beloaded into microcapsules or loaded freely in solution into the deviceor a combination thereof. Sample preparation may then be conducted, suchas by fragmenting the cDNA and attaching unique identifiers to thefragments. After sample preparation and recovery, the nucleic acidproducts of the reaction may be further analyzed, such as by sequencing.

Additionally, a device may be used to characterize multiple cellmarkers, similar to a flow cytometer. Any cell marker may becharacterized, including cell-surface markers (e.g., extracellularproteins, transmembrane markers) and markers located within the internalportion of a cell (e.g., RNA, mRNA, microRNA, multiple copies of genes,proteins, alternative splicing products, etc.). For example, cells maybe partitioned within the device, as described herein, so that at mostone cell is present within a microwell. Cell markers such as nucleicacids (e.g., RNA) may be extracted and/or fragmented prior to beinglabeled with a unique identifier (e.g., molecular bar code). Or,alternatively, the nucleic acids may be labeled with a unique identifierwithout being extracted and/or fragmented. The nucleic acids may then besubjected to further analysis such as sequencing reactions designed todetect multiple gene expression products. Such analysis may be useful ina number of fields. For example, if the starting cells are immune cells(e.g., T cells, B cells, macrophages, etc.), the analysis may provideinformation regarding multiple expressed markers and enableimmunophenotyping of the cells, for example by identifying different CDmarkers of the cells (e.g., CD3, CD4, CD8, CD19, CD20, CD 56, etc.).Such markers can provide insights into the function, character, class,or relative maturity of the cell. Such markers can also be used inconjunction with markers that are not necessarily immunophenotypingmarkers, such as markers of pathogenic infection (e.g., viral orbacterial protein, DNA, or RNA). In some cases, the device may be usedto identify at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 500, 700, 1000, 5000,10000, 50000, or 100000 different gene expression products or other formof cellular markers on a single-cell basis. Often, such methods do notcomprise use of dyes or probes (e.g., fluorescent probes or dyes).

Gene expression product analysis may be useful in numerous fieldsincluding immunology, cancer biology (e.g., to characterize theexistence, type, stage, aggressiveness, or other characteristic ofcancerous tissue), stem cell biology (e.g., in order to characterize thedifferentiation state of a stem cell, potency of a stem cell, cellulartype of a stem cell, or other features of a stem cell), microbiology,and others. The gene expression analysis may also be used in drugscreening applications, for example to evaluate the effect of aparticular drug or agent on the gene expression profile of particularcells.

VII. Terminology

The terminology used therein is for the purpose of describing particularembodiments only and is not intended to be limiting of a device of thisdisclosure. As used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. Furthermore, to the extent that the terms“including”, “includes”, “having”, “has”, “with”, or variants thereofare used in either the detailed description and/or the claims, suchterms are intended to be inclusive in a manner similar to the term“comprising”.

Several aspects of a device of this disclosure are described above withreference to example applications for illustration. It should beunderstood that numerous specific details, relationships, and methodsare set forth to provide a full understanding of a device. One havingordinary skill in the relevant art, however, will readily recognize thata device can be practiced without one or more of the specific details orwith other methods. This disclosure is not limited by the illustratedordering of acts or events, as some acts may occur in different ordersand/or concurrently with other acts or events. Furthermore, not allillustrated acts or events are required to implement a methodology inaccordance with this disclosure.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. The term “about” as used herein refers to a rangethat is 15% plus or minus from a stated numerical value within thecontext of the particular usage. For example, about 10 would include arange from 8.5 to 11.5.

The term microwell array, as used herein, generally refers to apredetermined spatial arrangement of microwells. Microwell array devicesthat comprise a microcapsule may also be referred to as “microwellcapsule arrays.” Further, the term “array” may be used herein to referto multiple arrays arranged on a surface, such as would be the casewhere a surface has multiple copies of an array. Such surfaces bearingmultiple arrays may also be referred to as “multiple arrays” or“repeating arrays.”

Example 1 Single Cell DNA Sequencing

A microwell capsule array is prepared to perform nucleic acid sequencingon individual human B-cells taken from a blood sample. Approximately15,000 cells are harvested and used for loading into the device. Adevice of this disclosure and containing 150,000 microwells is used.Each well is cylindrical in shape having a diameter of 125 um and aheight of 125 um, allowing at most 1 capsule to be loaded per well.Microcapsules made through emulsion polymerization with a PNIPAMhydrogel shell wall are created such that the microcapsules have adiameter of 100 um for loading in the device. The microcapsules arecreated such that the PNIPAM shell contains magnetic iron particles. Theouter surface of the shell is then chemically coupled to a antibodyspecific to a transmembrane B cell receptor on the outside of a B cell.

During the preparation process of capsules, reagents are simultaneouslyloaded into the capsules. Reagents necessary for cell lysis and labelingindividual DNA strands of the cells with DNA barcodes are loaded intocapsules. Reagents for cell lysis include a mild non-ionic detergent,buffer and salt. Reagents for the addition of DNA bar codes to genomicDNA included restriction enzymes, ligase, and >10,000,000 unique DNAoligonucleotides are loaded into capsules. Capsules are designed to besensitive to rupture at greater than 65 C.

Capsules are prepared to be applied to the microcapsule array. The arrayis placed on a magnetic temperature controlled hot plate. Microcapsulesare added to a sample of B cells such that one B cell is able to bind toone capsule. Capsule-cell conjugates are applied in aqueous carriersolution in a quantity in excess to the relative number of wells. Gentlepipetting of capsules-cells into the inlet port followed by applicationof a vacuum manifold to the outlet port distributes the capsulesthroughout the device. A magnetic field is applied through the plate.Excess capsule-cell solution is removed via pipetting through the outletport. Each capsule-cell conjugate is trapped and positioned inindividual wells via the magnetic field.

After the cells and capsules are loaded in the device, a carrier oil (orsealing fluid) is applied to the device to remove any excess aqueoussolution bridging adjacent microwells. The carrier oil applied to theinlet and excess oil is recovered at the outlet with a vacuum manifold.After the carrier oil is applied, the inlet and outlet ports are sealedwith tape.

The device is then heated, via the magnetic temperature controlled hotplate, to a temperature of 70 C for 10 min to allow for capsule ruptureand cell lysis. The hot plate is then switched to 37 C, for restrictionand ligation, for up to 1 hour.

After the sample preparation reaction is completed, the contents of thewells are recovered. The inlet and outlet ports of the device areunsealed and nitrogen gas is applied to the device to flush out theindividual components of the microwells. The sample is collected in bulkvia a pipette at the outlet port, while the magnetic field retainsruptured capsule shells in individual microwells.

The sample is then sequenced using a multiplex sequencing strategy knownin the art. Bar coding of individual cells allows for sequencinginformation to be gained for individual cells rather than as an averageof multiple cells. Based upon the number of cells sequenced and barcodes assigned, SNP cell-specific information is gained. Moreover, thenumber of reads for individual bar codes can be counted to provideinsight into the distribution of different types of cells with varyinggenetic backgrounds, within the original population of B cells.

Example 2 DNA Single Strand Sequencing

A microwell capsule array is prepared to perform nucleic acid sequencingon individual strands of DNA isolated from a population of human skincells. Cells are lysed using detergent and heat and approximately 15,000copies of diploid DNA are precipitated via chloroform/ethanolextraction. A resuspension of DNA is loaded into the device withapproximately 10,000 copies of haploid DNA. A device of this disclosure,with 300,000 microwells is used. Each well is cylindrical in shapehaving a diameter of 125 um and a height of 125 um, allowing at most 1capsule to be loaded per well. Microcapsules made through emulsionpolymerization with a PNIPAM hydrogel shell wall are created to aspecification of a sphere with a diameter of 100 um for loading into thedevice.

During the preparation of the microcapsules, reagents are simultaneouslyloaded into the capsules. The reagents include reagents necessary forlabeling individual DNA strands with DNA barcodes, including restrictionenzymes, ligase, and >10,000,000 unique DNA oligonucleotides. Capsulesdesigned to be sensitive to rupture at greater than 65 C are used forthe encapsulation.

Capsules are applied aqueous carrier solution in an excess to therelative number of wells. Gentle pipetting of capsules into the inletfollowed by application of a vacuum manifold to the outlet distributedthe capsules throughout the device. After excess capsule solution isremoved, a suspension of DNA in buffer is applied to the device in asimilar fashion as the capsules.

After the DNA strands and capsules are loaded in the device, a carrieroil is applied to the device to remove any excess aqueous solutionbridging adjacent microwells. The carrier oil is applied to the inletport and excess oil is recovered at the outlet port with a vacuummanifold. After the carrier oil is applied, the inlet and outlet portsare sealed with tape.

The device is then placed on a temperature controlled hot plate andheated to temperature of 70 C for 10 min to allow for capsule rupture.Reagents are released into the sample preparation reaction. The hotplate is then switched to 37 C, for restriction and ligation, for up to1 hour.

After the sample preparation reaction is completed, the inlet and outletports of the device are unsealed and nitrogen gas is applied to thedevice to flush out the individual components of the microwells. Thesample products, en bulk, are collected via pipette at the outlet port.

The sample is then sequenced to sufficient coverage (e.g., 500) using amultiplex sequencing strategy known in the art. Bar coding of individualDNA strands allows for sequencing information to be gained fromindividual strands rather than as an average of entire sample of DNA.Based upon the number of DNA strands sequenced and bar codes assigned,SNP phasing/haplotyping information is gained and many repetitiveregions of DNA can be resolved. In addition, a substantial boost inaccuracy can be gained by discarding mutations that appear randomly withrespect to haplotypes, as those are likely to be sequencing errors.

It should be understood from the foregoing that, while particularimplementations have been illustrated and described, variousmodifications may be made thereto and are contemplated herein. It isalso not intended that the invention be limited by the specific examplesprovided within the specification. While the invention has beendescribed with reference to the aforementioned specification, thedescriptions and illustrations of the preferable embodiments herein arenot meant to be construed in a limiting sense. Furthermore, it shall beunderstood that all aspects of the invention are not limited to thespecific depictions, configurations or relative proportions set forthherein which depend upon a variety of conditions and variables. Variousmodifications in form and detail of the embodiments of the inventionwill be apparent to a person skilled in the art. It is thereforecontemplated that the invention shall also cover any such modifications,variations and equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1.-77. (canceled)
 78. A method for generating a droplet, comprising: (a)combining a microcapsule comprising an oligonucleotide barcode and anucleic acid analyte each in an aqueous phase at a first junction of twoor more channels of a microfluidic device to form an aqueous mixturecomprising said microcapsule and said nucleic acid analyte, wherein saidmicrocapsule is degradable upon application of a stimulus; and (b)generating a droplet comprising said microcapsule and said nucleic acidanalyte by contacting said aqueous mixture with an immiscible continuousphase at a second junction of two or more channels of said microfluidicdevice.
 79. The method of claim 78, further comprising, in (a),combining said microcapsule, said nucleic acid analyte and one or morereagents necessary for amplification of said nucleic acid analyte atsaid first junction to form said aqueous mixture comprising saidmicrocapsule, said nucleic acid analyte and said one or more reagents.80. The method of claim 79, wherein in (b), said droplet furthercomprises said one or more reagents.
 81. The method of claim 80, whereinsaid one or more reagents comprises a polymerase.
 82. The method ofclaim 81, wherein said polymerase is unable to recognize uracil.
 83. Themethod of claim 78, wherein said microcapsule comprises a polymer gel.84. The method of claim 83, wherein said polymer gel is apolyacrylamide.
 85. The method of claim 78, wherein said microcapsulecomprises a bead.
 86. The method of claim 85, wherein said bead is a gelbead.
 87. The method of claim 78, wherein said microcapsule comprises achemical cross-linker.
 88. The method of claim 87, wherein said chemicalcross-linker is a disulfide bond.
 89. The method of claim 78, whereinsaid stimulus is selected from the group consisting of a biologicalstimulus, a chemical stimulus, a thermal stimulus, an electricalstimulus, a magnetic stimulus, a photo stimulus, and a combinationthereof.
 90. The method of claim 89, wherein said chemical stimulus isselected from the group consisting of a change in pH, change in ionconcentration, and a reducing agent.
 91. The method of claim 78,wherein, during or after (a), said stimulus is a chemical stimulus andsaid chemical stimulus is contacted with said microcapsule.
 92. Themethod of claim 78, wherein a nucleic acid molecule comprises saidoligonucleotide barcode and wherein said nucleic acid comprises uracil.93. The method of claim 78, wherein said microcapsule comprises a primerthat comprises said oligonucleotide barcode.
 94. The method of claim 93,wherein said primer further comprises a random N-mer.
 95. The method ofclaim 93, further comprising, after (b), amplifying said nucleic acidanalyte with said primer.
 96. The method of claim 78, wherein saidnucleic acid analyte is selected from the group consisting of DNA, RNA,dNTPs, ddNTPs, amplicons, synthetic nucleotides, syntheticpolynucleotides, polynucleotides, oligonucleotides, peptide nucleicacids, cDNA, dsDNA, ssDNA, plasmid DNA, cosmid DNA, High MolecularWeight (MW) DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA,mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA,scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA.
 97. Themethod of claim 78, wherein said oligonucleotide barcode is coupled tosaid microcapsule via a chemical cross-linker.
 98. The method of claim78, wherein said oligonucleotide barcode is coupled to said microcapsulevia a disulfide bond.
 99. The method of claim 78, wherein saidoligonucleotide barcode is coupled to said microcapsule via a covalentbond.
 100. The method of claim 78, wherein said oligonucleotide barcodeis coupled to said microcapsule via a labile moiety.
 101. The method ofclaim 78, wherein said oligonucleotide barcode is reversibly immobilizedto said microcapsule.